Method for refining vasopressin

Abstract

A method for refining vasopressin, including: subjecting a crude vasopressin solution to reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification sequentially using reversed-phase high performance liquid chromatography. The crude vasopressin solution is obtained by oxidizing a crude reduced vasopressin prepared by solid phase synthesis. A super water-resistant packing material is used in the reversed-phase high performance liquid chromatography.

What is claimed is: 
     
         1 . A method for refining vasopressin, comprising:
 subjecting a crude vasopressin solution to reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification sequentially using reversed-phase high performance liquid chromatography (RP-HPLC); wherein a packing material used in the RP-HPLC is a water-resistant packing material; the reversed-phase enrichment, the reversed-phase salt conversion and the reversed-phase purification are all completed in one reversed-phase elution, and conditions of the reversed-phase elution are listed as follows:   
       
         
           
                 
                 
                 
               
                     
                 
                   Steps 
                   Time 
                   Eluent 
                 
                     
                 
                     
                 
                 
                 
                 
                 
               
                   1 
                   0-50 
                   min 
                   100% sample C1 
                 
                   2 
                   51-71 
                   min 
                   100% mobile phase C2 
                 
                   3 
                   72-90 
                   min 
                   100% mobile phase A 
                 
                   4 
                   90-95 
                   min 
                   100% mobile phase A→90% mobile phase 
                 
                     
                     
                     
                   A + 10% mobile phase B 
                 
                   5 
                   95-125 
                   min 
                   90% mobile phase A + 10% mobile phase 
                 
                     
                     
                     
                   B→80% mobile phase A + 20% mobile 
                 
                     
                     
                     
                   phase B 
                 
                     
                 
             
                
                
                
               
               
                
               
            
             
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       wherein the mobile phase A consists of 0.005-0.1% by volume of acetic acid and water; the mobile phase B consists of 0.005-0.1% by volume of acetic acid and acetonitrile; the sample C1 is the crude vasopressin solution; the mobile phase C2 is a 5-50 mM aqueous NH 4 Ac—NH 4 OH solution at pH 7.0-9.0; and a flow rate of the eluent is 80-100 mL/min; and
 collecting an eluate within a retention time of 105-115 min to obtain a pure vasopressin solution. 
 
     
     
         2 . The method of  claim 1 , wherein the crude vasopressin solution is obtained by dissolving, diluting, and oxidizing a crude reduced vasopressin product prepared by solid phase synthesis. 
     
     
         3 . The method of  claim 2 , wherein the crude reduced vasopressin product is dissolved and diluted to produce a solution of the crude reduced vasopressin product; a concentration of the solution of the crude reduced vasopressin product is 0.1-4 mg/mL; and a solvent for dissolving the crude reduced vasopressin product is a 50% aqueous acetic acid solution. 
     
     
         4 . The method of  claim 3 , wherein the concentration of the solution of the crude reduced vasopressin product is 0.5-2 mg/mL. 
     
     
         5 . The method of  claim 1 , wherein a formula of vasopressin in the crude vasopressin solution is 
       
         
           
           
               
               
           
         
       
       and a solvent of the crude vasopressin solution is an aqueous solution containing trifluoroacetic acid and acetic acid. 
     
     
         6 . The method of  claim 1 , wherein the water-resistant packing material is UniSil® ODS-AQ material. 
     
     
         7 . The method of  claim 1 , wherein the water-resistant packing material has a pore diameter of 7-10 nm and a particle size of 10 μm. 
     
     
         8 . The method of  claim 1 , wherein a detection wavelength of the RP-HPLC is 220 nm. 
     
     
         9 . The method of  claim 1 , wherein the mobile phase A consists of 0.02-0.05% by volume of acetic acid and water; and/or
 the mobile phase B consists of 0.02-0.05% by volume of acetic acid and acetonitrile; and/or   the mobile phase C2 is a 10-20 mM aqueous NH 4 Ac—NH 4 OH solution; and/or   the pH of the mobile phase C2 is 7.5-8.5; and/or   a HPLC purity of crude vasopressin in the crude vasopressin solution is 60%-85%.   
     
     
         10 . The method of  claim 9 , wherein the HPLC purity of the crude vasopressin in the crude vasopressin solution is 70%-85%. 
     
     
         11 . The method of  claim 10 , wherein the HPLC purity of the crude vasopressin in the crude vasopressin solution is 70%-80%. 
     
     
         12 . The method of  claim 1 , wherein during a period of 50-51 min, the eluent is changed from the sample C1 to the mobile phase C2; and during a period of 71-72 min, the eluent is changed from the mobile phase C2 to the mobile phase A. 
     
     
         13 . The method of  claim 1 , wherein in steps (4) and (5) of the reversed-phase elution, a proportion of the mobile phase A in the eluent decreases at a uniform rate and a proportion of the mobile phase B in the eluent increases at a uniform rate.